DESCRIPTION(Adapted from applicant's abstract): Arachidonylethanolamide (AEA, "anandamide") is a constituent lipid in brain that activates the CB1 cannabinoid receptor. While a growing body of evidence supports the hypothesis that AEA is an agonist of the CB1 receptor in vitro,many fundamental questions regarding the role of AEA in the brain remain unanswered. The overall hypothesis of this application is that an AEA/CB 1 signaling system operates in the brain. Furthermore, we hypothesize that three proteins involved in this signaling system: phospholipase D (PLD); an AEA transport protein; and fatty acyl amide hydrolase (FAAH) are subject to regulation by signaling cascades in neurons. These experiments will begin to explore the integration of the endocannabinoids into signaling processes in the brain. The following specif aboutc aims will be used to test this hypothesis. (1.) The phospholipase D (PLD) involved in AEA synthesis will be characterized. We will design and synthesize inhibitors and test their activity and specificity. We will detennine whether the relatively well characterized isoforms of PLD (PLDI and PLD2) synthesis AE4 from N-arachidonylPE; (2.) AEA is moved across cell membranes by a protein carrier. We will determine whether the driving force for AEA accumulation by cells is sequestration of AEA by an intracellular protein or membrane lipid or catabolism of AEA intracellularly. We will optimize inhibitors of the AEA carrier. (3.) Fatty acid arnide hydrolase (FAAH) is a microsomal enzyme that hydrolyzes AEA. We will elucidate the active site of FAAH using an inhibitor that covalently bonds to active site residues. We will deterrnine whether FAAH associates with other cellular proteins via an SH3 binding domain. This project is a collaboration among scientists with expertise in lipid chemistry and biochemistry; synthetic and analytical chemistry of arachidonic acid analogs; biochemical enzymology and cannabinoid receptor pharmacology. We anticipate that successful completion of these studies will provide valuable insight into the role of the AEA/CB 1 signaling system in the brain.